Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ∼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

The exquisite selectivity, sensitivity, and spatial resolution obtained with fluorescence spectroscopy and imaging have led to an ever-increasing number of applications. With the development of detectors approaching 100 % quantum efficiencies and sophisticated collection optics, the bottleneck of current fluorescence microscopy is the fluorophores used, which pose severe limitations owing to photobleaching and blinking. Most of the basic dye structures that are currently used in fluorescence microscopy have been known since their use in the development of dye lasers.[1] Increasing demands posed by fluorescence microscopy and single-molecule and high-resolution applications [2,3] have spurred the development of new kinds of emitters such as semiconductor nanocrystals, silver nanoclusters, and new derivatives of fluorescent proteins.[4] In comparison, the advancement of classical organic dyes such as rhodamine or cyanine derivatives has been incremental despite some progress with regard to labeling chemistry, solubility in water, and the availability of bright and photostable near-IR dyes. Approaches for their improvement comprise increasing brightness by multichromophore systems, intramolecular triplet quenching, and decreasing the sensibility for reactions with singlet oxygen. [5] For different reasons, none of these approaches has been implemented with great success in fluorescence microscopy.Here we present a new approach to minimize photobleaching and blinking by recovering reactive intermediates. The method is based on the removal of oxygen and quenching of triplet as well as charge-separated states by electrontransfer reactions. For this reason, a structure that contains reducing as well as oxidizing agents, that is, a reducing and oxidizing system (ROXS) is used. The success of the approach is demonstrated by single-molecule fluorescence spectroscopy of oligonucleotides labeled with different fluorophores, that is, cyanines, (carbo-)rhodamines, and oxazines, in aqueous solvents; individual fluorophores can be observed for minutes under moderate excitation with increased fluorescence brightness. Thermodynamic considerations of the underlying redox reactions support the model, yielding a comprehensive picture of blinking and photobleaching of organic fluorophores.Typically, the photophysics of fluorophores is described by a three-state model including the ground and first excited singlet states, S 0 and S 1 , respectively, and the lowest triplet state T 1 . Owing to its longer lifetime, T 1 is considered to be the photochemically most active state. Quenching of T 1 by molecular oxygen, for example, can generate reactive singlet oxygen, and therefore oxygen is removed in demanding applications, for example, with the aid of an enzymatic oxygen-scavenging system.[6] The disadvantage of oxygen removal, however, is the increase of the triplet state lifetime with negative effects for the brightness of the fluorophore and increased probability for other follow-up reactions from the triplet state. Alternatively, redu...

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm−2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm−2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence lifetime of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.

We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher beta-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of >90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.

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The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.